As a potent and specific inhibitor of gene expression, siRNA is being rapidly adopted as the preferred tool for functional genomics research [3, 4]. siRNA oligos are typically used to inhibit an individual gene, though targeting multiple genes or groups of genes are possible by using a combination of multiple siRNA sequences [5, 6]. The success of using siRNA to knockdown gene expression in vitro has led to a growing interest in applications of siRNA inhibitors in vivo for evaluation of the therapeutic potentials of the gene targets of interest, potency of siRNA inhibitors, the route of administration, and the unwanted adverse effects. These applications should eventually provide validated targets for conventional therapeutic modalities such as small molecule and monoclonal antibody inhibitors, as well as validation of siRNA drug lead itself (Fig. 3.1) [3].
One example is using siRNA oligos specifically targeting angiogenesis factors, such as VEGFs, EGF, FGFs, and their receptors, representing the most widely recognized targets that took years to validate. One study using siRNA-mediated downregulation of these proangiogenesis genes, VEGF and VEGF R2, in clinically relevant xenograft tumor models resulted in a significant antitumor efficacy. Thus, the functions of these two targets were further validated rapidly in a matter of weeks [7]. This example demonstrates the power of in vivo target validation with
Preclinical Pharm & Tox ADME, CMC
Clinical Studies:
Final Validation Lead
Discovery Target
Validation In vivo Target ID &
Validation In vitro
Successful Drug
Conventional Drugs:
Antibiotics, small molecules and monoclonal antibodies RNAi Therapeutics
Designed siRNA
Validated siRNA Validated
Target
Validated Delivery
Fig. 3.1 Delivery of siRNA for drug discovery and development. Effective siRNA deliveries in vitro and in vivo are playing very important role for siRNA-based target validation and potential RNAi therapeutics [5]. When targets are validated through in vivo siRNA delivery process, there are already three types of outcomes: validated targets, validated siRNA duplexes, and validated in vivo delivery
siRNA inhibitors. In this case, not only the roles of the proangiogenic factors were validated, the siRNA inhibitors themselves were also validated as potential anticancer drugs.
Delivering siRNA oligos in vivo to animal tissues and keeping them active in the targeted cells are complicated and involve using a physical, chemical, or biological approach and in some cases their combination [7]. Since the main goal of in vivo delivery is to have active siRNA oligos in the target cells, the stability of siRNA oligos in both the extracellular and intracellular environment after a systemic administration is the most challenging issue (Fig. 3.2). The first hurdle is the size of the 21–23-nt double-stranded siRNA oligos: they are relatively small and thus rapidly excreted through urine when administrated into the blood stream, even if those siRNA molecules remain stable through chemical modifications.
Second, the double-stranded siRNA oligos are relatively unstable in the serum environment and are potentially degraded by RNase activity within a short period of time. Third, when siRNA is administered systemically, the nonspecific dis- tribution of the oligos throughout the body will significantly decrease the local concentration at the site of disease. In addition, the siRNA oligos need to overcome the blood vessel endothelial wall and multiple tissue barriers in order to reach the target cells. Finally, when siRNA reaches the target cells, cellular uptake of the oligos and intracellular RNAi activity require efficient endocytosis and intact double- stranded oligos.
Cytoplasm mRNA siRNA
RISC
Nucleus Gene Silencing Serum
Instability
Tissue Barricades
Non-specific Distribution Excretion
Hurdles to siRNA
systemic delivery Potent cellular
activity
Fig. 3.2 Challenges of systemic in vivo siRNA delivery. The in vivo application, especially systemic delivery of siRNA, is facing challenges from multiple hurdles in the extracellular environment and various barriers for the intracellular uptake. Addressing those issues is critical for efficient in vivo delivery of siRNA in preclinical animal models for drug target validation and potential therapeutics
To increase their stability in both extracellular and intracellular environments, siRNA oligos can be chemically modified by a variety of methods, including change of oligo backbone, replacement of individual nucleotide with nucleotide analogue, and adding conjugates to the oligo. The chemically modified siRNA demonstrated a significant serum resistance and higher stability [8], but it did not solve the problems of excretion through urine and targeted delivery. Therefore, a delivery system capable of protecting siRNA oligos from the urinary excretion and RNase degradation, transporting siRNA oligos through the physical barriers to the target tissue, and enhancing cellular uptake of the siRNA, is the key to the success of in vivo siRNA application.
The accessibility of different tissue types, the presence of various delivery routes, and a variety of pharmacological requirements makes it impossible to have a universal in vivo delivery system suitable to every scenario of siRNA delivery. In terms of in vivo delivery vehicles for siRNA, the “nonviral” carriers are the major type being investigated so far, though some physical and viral delivery approaches are also very effective. The routes of in vivo deliveries are commonly categorized as local or systemic. Some of the delivery vehicles and delivery routes are very effective in animals for target validation but may not be useful for delivery of siRNA therapeutics in humans (Fig. 3.3). Therefore, in vivo siRNA delivery carriers and methods can also be classified as clinically viable and nonclinically viable, according to their suitability for the human use.
Disease Models:
Xenograft Tumor Ocular Neovasculature Respiratory Inflammation Collagen Induced Arthritis CNS Diseases
Readouts:
Pharmacology Pathology Efficacy and Toxicity
Applications:
Cancer AMD, Retinopathy Asthma, COPD, SARS Rheumatoid Arthritis Parkinson Disease Delivery Routes
siRNA Naked Conjugated Modified Liposome Carrier Polymer Carrier
Ligand-Targeted Nanoparticle
Intra-Articular Intra-Tumor
Intra-Muscle Intra-Tracheal
Intra-Vitreous Intra-Nasal
Intra-Venous Intra-Peritoneal Local
Systemic
Intra-Cerebral
Fig. 3.3 Applications of in vivo siRNA delivery in disease models. Mouse models are widely used for in vivo siRNA delivery studies. siRNA can be delivered by many routes based on the disease types and targeted tissues. The efficacy and toxicity readouts of the siRNA inhibitors from the preclinical models will provide vital information for the in vivo target validation [9]. The clinically viable siRNA delivery provides foundation for designing the administration route and condition of an RNAi therapeutic protocol